Method of diagnosing, monitoring, and staging colon cancer

ABSTRACT

The present invention provides a new method for detecting, diagnosing, monitoring, staging, and prognosticating colon cancer.

This application claims benefit of U.S. Provisional No. 60/086,266 filedMay 21, 1998.

FIELD OF THE INVENTION

This invention relates, in part, to newly developed assays fordetecting, diagnosing, monitoring, staging, and prognosticating cancers,particularly colon cancer.

BACKGROUND OF THE INVENTION

Colon cancer is the second most frequently diagnosed malignancy in theUnited States. Cancer of the gastrointestinal tract, especially coloncancer, is a highly treatable and often a curable disease when localizedto the bowel. However, currently colon cancer is the second most commoncause of cancer death. Surgery is the primary treatment and results incure in approximately 50% of patients. Recurrence following surgery is amajor problem and often is the ultimate cause of death. The prognosis ofcolon cancer is clearly related to the degree of penetration of thetumor through the bowel wall and the presence or absence of nodalinvolvement. These two characteristics form the basis for all stagingsystems developed for this disease. Bowel obstruction and bowelperforation are indicators of poor prognosis. Elevated pretreatmentserum levels of carcinoembryonic antigen (CEA) and carbohydrate antigen19-9 (CA 19-9) also have negative prognostic significance.

Because of the frequency of the disease (approximately 160,000 new casesof colon cancer per year), the identification of high-risk groups, thedemonstrated slow growth of primary lesions, the better survival ofearly-stage lesions, and the relative simplicity and accuracy ofscreening tests, screening for colon cancer should be a part of routinecare for all adults starting at age 50, especially those withfirst-degree relatives with colorectal cancer.

Procedures used for detecting, diagnosing, monitoring, staging, andprognosticating colon cancer are of critical importance to the outcomeof the patient. For example, patients diagnosed with early colon cancergenerally have a much greater five-year survival rate as compared to thesurvival rate for patients diagnosed with distant metastasized coloncancer. Treatment decisions are usually made in reference to the olderDukes or the Modified Astler-Coller (MAC) classification schema forstaging. However, new diagnostic methods which are more sensitive andspecific for detecting early colon cancer are clearly needed.

Further, colon cancer patients must be closely monitored followinginitial therapy and during adjuvant therapy to determine response totherapy and to detect persistent or recurrent disease of metastasis.Thus, there is clearly a need for a colon cancer marker which is moresensitive and specific in detecting colon cancer recurrence.

Another important step in managing colon cancer is to determine thestage of the patient's disease. Stage determination has potentialprognostic value and provides criteria for designing optimal therapy.Currently, pathological staging of colon cancer is preferable overclinical staging as pathological staging provides a more accurateprognosis. However, clinical staging would be preferred were the methodof clinical staging at least as accurate as pathological staging becauseit does not depend on an invasive procedure to obtain tissue forpathological evaluation. Staging of colon cancer would be improved bydetecting new markers in cells, tissues, or bodily fluids which coulddifferentiate between different stages of invasion.

In the present invention, methods are provided for detecting,diagnosing, monitoring, staging, and prognosticating colon cancers,particularly colon, stomach, and small intestine cancer, via nine (9)Colon Specific Genes (CSGs). The nine CSGs refer, among other things, tonative proteins expressed by the genes comprising the polynucleotidesequences of any of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8 or 9. In thealternative, what is meant by the nine CSGs as used herein, means thenative mRNAs encoded by the genes comprising any of the polynucleotidesequences of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8 or 9 or levels of thegenes comprising any of the polynucleotide sequences of SEQ ID NO:1, 2,3, 4, 5, 6, 7, 8 or 9.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill in the art from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide a method for diagnosing the presence of colon cancer in apatient which comprises measuring levels of CSG in a sample of cells,tissue or bodily fluid from the patient and comparing the measuredlevels of CSG with levels of CSG in preferably the same cells, tissue,or bodily fluid type of a control, wherein an increase in the measuredCSG levels in the patient versus levels of CSG in the control isassociated with colon cancer.

Another object of the present invention is to provide a method ofdiagnosing metastatic colon cancer in a patient which comprisesmeasuring CSG levels in a sample of cells, tissue, or bodily fluid fromthe patient and comparing the measured CSG levels with levels of CSG inpreferably the same cells, tissue, or bodily fluid type of a control,wherein an increase in measured CSG levels in the patient versus levelsof CSG in the control is associated with a cancer which hasmetastasized.

Another object of the present invention is to provide a method ofstaging colon cancer in a patient which comprises identifying a patienthaving colon cancer, measuring levels of CSG in a sample of cells,tissues, or bodily fluid obtained from the patient, and comparing themeasured CSG levels with levels of CSG in preferably the same cells,tissue or bodily fluid type of a control. An increase in measured CSGlevels in the patient versus CSG levels in the control can be associatedwith a cancer which is progressing while a decrease or equivalent levelof CSG measured in the patient versus the control can be associated witha cancer which is regressing or in remission.

Another object of the present invention is to provide a method ofmonitoring colon cancer in a patient for the onset of metastasis. Themethod comprises identifying a patient having colon cancer that is notknown to have metastasized, periodically measuring levels of CSG in asample of cells, tissues, or bodily fluid obtained from the patient, andcomparing the measured CSG levels with levels of CSG in preferably thesame cells, tissue, or bodily fluid type of a control, wherein anincrease in measured CSG levels versus control CSG levels is associatedwith a cancer which has metastasized.

Yet another object of the present invention is to provide a method ofmonitoring the change in stage of colon cancer in a patient whichcomprises identifying a patient having colon cancer, periodicallymeasuring levels of CSG in a sample of cells, tissue, or bodily fluidobtained from the patient, and comparing the measured CSG levels withlevels of CSG in preferably the same cells, tissues, or bodily fluidtype of a control wherein an increase in measured CSG levels versus thecontrol CSG levels is associated with a cancer which is progressing anda decrease in the measured CSG levels versus the control CSG levels isassociated with a cancer which is regressing or in remission.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill in the art from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The present invention relates to diagnostic assays and methods, bothquantitative and qualitative for detecting, diagnosing, monitoring,staging, and prognosticating cancers by comparing levels of CSG withthose of CSG in a normal human control. What is meant by “levels of CSG”as used herein, means levels of the native protein expressed by thegenes comprising the polynucleotide sequence of any of SEQ ID NO: 1, 2,3, 4, 5, 6, 7, 8 or 9. In the alternative, what is meant by “levels ofCSG” as used herein, means levels of the native mRNA encoded by any ofthe genes comprising any of the polynucleotide sequences of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8 or 9 or levels of the gene comprising any of thepolynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9. Suchlevels are preferably measured in at least one of, cells, tissues and/orbodily fluids, including determination of normal and abnormal levels.Thus, for instance, a diagnostic assay in accordance with the inventionfor diagnosing over-expression of any one of the CSG proteins comparedto normal control bodily fluids, cells, or tissue samples may be used todiagnose the presence of cancers, including colon cancer. Any of thenine CSGs may be measured alone in the methods of the invention, or alltogether or any combination of the nine.

By “control” it is meant a human patient without cancer and/or noncancerous samples from the patient, also referred to herein as a normalhuman control; in the methods for diagnosing or monitoring formetastasis, control may also include samples from a human patient thatis determined by reliable methods to have colon cancer which has notmetastasized.

All the methods of the present invention may optionally includemeasuring the levels of other cancer markers as well as CSG. Othercancer markers, in addition to CSG, useful in the present invention willdepend on the cancer being tested and are known to those of skill in theart.

Diagnostic Assays

The present invention provides methods for diagnosing the presence ofcolon cancer by analyzing for changes in levels of CSG in cells, tissuesor bodily fluids compared with levels of CSG in cells, tissues or bodilyfluids of preferably the same type from a normal human control, whereinan increase in levels of CSG in the patient versus the normal humancontrol is associated with the presence of colon cancer. Withoutlimiting the instant invention, typically, for a quantitative diagnosticassay a positive result indicating the patient being rested has canceris one in which cells, tissues, or bodily fluid levels of the cancermarker, such as CSG, are at least two times higher, and most preferablyare at least five times higher, than in preferably the same cells,tissues, or bodily fluid of a normal human control.

The present invention also provides a method of diagnosing metastaticcolon cancer in a patient having colon cancer which has not yetmetastasized for the onset of metastasis. In the method of the presentinvention, a human cancer patient suspected of having colon cancer whichmay have metastasized (but which was not previously known to havemetastasized) is identified. This is accomplished by a variety of meansknown to those of skill in the art. For example, in the case of coloncancer, patients are typically diagnosed with colon cancer followingtraditional detection methods.

In the present invention, determining the presence of CSG level incells, tissues, or bodily fluid, is particularly useful fordiscriminating between colon cancer which has not metastasized and coloncancer which has metastasized. Existing techniques have difficultydiscriminating between colon cancer which has metastasized and coloncancer which has not metastasized and proper treatment selection isoften dependent upon such knowledge.

In the present invention, the cancer marker levels measured in suchcells, tissues, or bodily fluid is CSG, and are compared with levels ofCSG in preferably the same cells, tissue, or bodily fluid type of anormal human control. That is, if the cancer marker being observed isjust CSG in serum, this level is preferably compared with the level ofCSG in serum of a normal human patient. An increase in the CSG in thepatient versus the normal human control is associated with colon cancerwhich has metastasized.

Without limiting the instant invention, typically, for a quantitativediagnostic assay a positive result indicating the cancer in the patientbeing tested or monitored has metastasized is one in which cells,tissues, or bodily fluid levels of the cancer marker, such as CSG, areat least two times higher, and most preferable are at least five timeshigher, than in preferably the same cells, tissues, or bodily fluid of anormal patient.

Normal human control as used herein includes a human patient withoutcancer and/or non cancerous samples from the patient; in the methods fordiagnosing or monitoring for metastasis, normal human control may alsoinclude samples from a human patient that is determined by reliablemethods to have colon cancer which has not metastasized.

Staging

The invention also provides a method of staging colon cancer in a humanpatient.

The method comprises identifying a human patient having such cancer;analyzing a sample of cells, tissues, or bodily fluid from such patientfor CSG. Then, the method compares CSG levels in such cells, tissues, orbodily fluid with levels of CSG in preferably the same cells, tissues,or bodily fluid type of a normal human control sample, wherein anincrease in CSG levels in the patient versus the normal human control isassociated with a cancer which is progressing and a decrease in thelevels of CSG is associated with a cancer which is regressing or inremission.

Monitoring

Further provided is a method of monitoring colon cancer in a humanhaving such cancer for the onset of metastasis. The method comprisesidentifying a human patient having such cancer that is not known to havemetastasized; periodically analyzing a sample of cells, tissues, orbodily fluid from such patient for CSG; comparing the CSG levels in suchcells, tissue, or bodily fluid with levels of CSG in preferably the samecells, tissues, or bodily fluid type of a normal human control sample,wherein an increase in CSG levels in the patient versus the normal humancontrol is associated with a cancer which has metastasized.

Further provided by this inventions is a method of monitoring the changein stage of colon cancer in a human having such cancer. The methodcomprises identifying a human patient having such cancer; periodicallyanalyzing a sample of cells, tissues, or bodily fluid from such patientfor CSG; comparing the CSG levels in such cells, tissue, or bodily fluidwith levels of CSG in preferably the same cells, tissues, or bodilyfluid type of a normal human control sample, wherein an increase in CSGlevels in the patient versus the normal human control is associated witha cancer which is progressing in stage and a decrease in the levels ofCSG is associated with a cancer which is regressing in stage or inremission.

Monitoring such patient for onset of metastasis is periodic andpreferably done on a quarterly basis. However, this may be more or lessfrequent depending on the cancer, the particular patient, and the stageof the cancer.

Assay Techniques

Assay techniques that can be used to determine levels of geneexpression, such as CSG of the present invention, in a sample derivedfrom a host are well-known to those of skill in the art. Such assaymethods include radioimmunoassays, reverse transcriptase PCR (RT-PCR)assays, immunohistochemistry assays, in situ hybridization assays,competitive-binding assays, Western Blot analyses and ELISA assays.Among these, ELISAs are frequently preferred to diagnose a gene'sexpressed protein in biological fluids. An ELISA assay initiallycomprises preparing an antibody, if not readily available from acommercial source, specific to CSG, preferably a monoclonal antibody. Inaddition a reporter antibody generally is prepared which bindsspecifically to CSG. The reporter antibody is attached to a detectablereagent such as radioactive, fluorescent or enzymatic reagent, forexample horseradish peroxidase enzyme or alkaline phosphatase.

To carry out the ELISA, antibody specific to CSG is incubated on a solidsupport, e.g., a polystyrene dish, that binds the antibody. Any freeprotein binding sites on the dish are then covered by incubating with anon-specific protein such as bovine serum albumin. Next, the sample tobe analyzed is incubated in the dish, during which time CSG binds to thespecific antibody attached to the polystyrene dish. Unbound sample iswashed out with buffer. A reporter antibody specifically directed to CSGand linked to horseradish peroxidase is placed in the dish resulting inbinding of the reporter antibody to any monoclonal antibody bound toCSG. Unattached reporter antibody is then washed out. Reagents forperoxidase activity, including a calorimetric substrate are then addedto the dish. Immobilized peroxidase, linked to CSG antibodies, producesa colored reaction product. The amount of color developed in a giventime period is proportional to the amount of CSG protein present in thesample. Quantitative results typically are obtained by reference to astandard curve.

A competition assay may be employed wherein antibodies specific to CSGattached to a solid support and labeled CSG and a sample derived fromthe host are passed over the solid support and the amount of labeldetected attached to the solid support can be correlated to a quantityof CSG in the sample. Nucleic acid methods may be used to detect CSGmRNA as a marker for colon cancer. Polymerase chain reaction (PCR) andother nucleic acid methods, such as ligase chain reaction (LCR) andnucleic acid sequence based amplification (NASABA), can be used todetect malignant cells for diagnosis and monitoring of variousmalignancies. For example, reverse-transcriptase PCR (RT-PCR) is apowerful technique which can be used to detect the presence of aspecific mRNA population in a complex mixture of thousands of other mRNAspecies. In RT-PCR, an mRNA species is first reverse transcribed tocomplementary DNA (cDNA) with use of the enzyme reverse transcriptase;the cDNA is then amplified as in a standard PCR reaction. RT-PCR canthus reveal by amplification the presence of a single species of mRNA.Accordingly, if the mRNA is highly specific for the cell that producesit, RT-PCR can be used to identify the presence of a specific type ofcell.

Hybridization to clones or oligonucleotides arrayed on a solid support(i.e., gridding) can be used to both detect the expression of andquantitate the level of expression of that gene. In this approach, acDNA encoding the CSG gene is fixed to a substrate. The substrate may beof any suitable type including but not limited to glass, nitrocellulose,nylon or plastic. At least a portion of the DNA encoding the CSG gene isattached to the substrate and then incubated with the analyte, which maybe RNA or a complementary DNA (cDNA) copy of the RNA, isolated from thetissue of interest.

Hybridization between the substrate bound DNA and the analyte can bedetected and quantitated by several means including but not limited toradioactive labeling or fluorescence labeling of the analyte or asecondary molecule designed to detect the hybrid. Quantitation of thelevel of gene expression can be done by comparison of the intensity ofthe signal from the analyte compared with that determined from knownstandards. The standards can be obtained by in vitro transcription ofthe target gene, quantitating the yield, and then using that material togenerate a standard curve.The above tests can be carried out on samples derived from a variety ofpatients' cells, bodily fluids and/or tissue extracts (homogenates orsolubilized tissue) such as from tissue biopsy and autopsy material.Bodily fluids useful in the present invention include blood, urine,saliva, or any other bodily secretion or derivative thereof. Blood caninclude whole blood, plasma, serum, or any derivative of blood.

EXAMPLES

The present invention is further described by the following examples.These examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

Example 1 CSGs

Searches were carried out and CSGs identified using the following SearchTools as part of the LIFESEQ® database available from IncytePharmaceuticals, Palo Alto, Calif.:

1. Library Comparison (compares one library to one other library) allowsthe identification of clones expressed in tumor and absent or expressedat a lower level in normal tissue.

2. Subsetting is similar to library comparison but allows theidentification of clones expressed in a pool of libraries and absent orexpressed at a lower level in a second pool of libraries.

3. Transcript Imaging lists all of the clones in a single library or apool of libraries based on abundance. Individual clones can then beexamined using Electronic Northerns to determine the tissue sources oftheir component ESTs.

4. Protein Function: Incyte has identified subsets of ESTs with apotential protein function based on homologies to known proteins. Someexamples in this database include Transcription Factors and Proteases.We identified some leads by searching in this database for clones whosecomponent ESTs showed disease specificity.

Electronic subtractions, transcript imaging and protein functionsearches were used to identify clones, whose component ESTs wereexclusively or more frequently found in libraries from specific tumors.Individual candidate clones were examined in detail by checking whereeach EST originated.

TABLE 1 CSGs SEQ ID NO: Clone ID # Gene ID # 1 238330 242807 TranscriptImaging 2 1285234 239588 Subsetting 3 1341701 29634 Transcript Imaging 4816257 233421 Subsetting 5 775133 245080 Subsetting 6 1335450 245811Subsetting 7 2348122 233711 Transcript Imaging 8 3228674 230273Subsetting 9 1632174 229022 Transcript Imaging

The following example was carried out using standard techniques, whichare well known and routine to those of skill in the art, except whereotherwise described in detail. Routine molecular biology techniques ofthe following example can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Example 2 Relative Quantitation of CSG Gene Expression

Real-Time quantitative PCR with fluorescent Taqman probes is aquantitation detection system utilizing the 5′-3′ nuclease activity ofTaq DNA polymerase. The method uses an internal fluorescentoligonucleotide probe (Taqman) labeled with a 5′ reporter dye and adownstream, 3′ quencher dye. During PCR, the 5′-3′ nuclease activity ofTaq DNA polymerase releases the reporter, whose fluorescence can then bedetected by the laser detector of the Model 7700 Sequence DetectionSystem (PE Applied Biosystems, Foster City, Calif., USA).

Amplification of an endogenous control is used to standardize the amountof sample RNA added to the reaction and normalize for ReverseTranscriptase (RT) efficiency. Either cyclophilin,glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S ribosomal RNA(rRNA) is used as this endogenous control. To calculate relativequantitation between all the samples studied, the target RNA levels forone sample are used as the basis for comparative results (calibrator).Quantitation relative to the “calibrator” can be obtained using thestandard curve method or the comparative method (User Bulletin #2: ABIPRISM 7700 Sequence Detection System).

To evaluate the tissue distribution, and the level of CSGs in normal andtumor tissue, total RNA was extracted from normal tissues, tumortissues, and from tumors and the corresponding matched normal tissues.Subsequently, first strand cDNA was prepared with reverse transcriptaseand the polymerase chain reaction was done using primers and Taqmanprobe specific to the CSG. The results were analyzed using the ABI PRISM7700 Sequence Detector. The absolute numbers are relative levels ofexpression of the CSG compared to the calibrator.

Comparative Examples

Similar mRNA expression analysis for genes coding for the diagnosticmarkers PSA (Prostate Specific Antigen) and PLA2 (Phospholipase A2) wasperformed for comparison. PSA is currently the only cancer screeningmarker available in clinical laboratories. When the panel of normalpooled tissues was analyzed, PSA was expressed at very high levels inprostate, with a very low expression in breast and testis. Afteranalysis of more than 55 matching samples from 14 different tissues, thedata corroborated the tissue specificity seen with normal tissuesamples. PSA expression was compared in cancer and normal adjacenttissue for 12 matching samples of prostate tissue. The relative levelsof PSA were higher in 10 cancer samples (83%). Clinical data recentlyobtained support the utilization of PLA2 as a staging marker for latestages of prostate cancer. mRNA expression data described herein showedoverexpression of the mRNA in 8 out of the 12 prostate matching samplesanalyzed (66%). PLA2 had high levels of mRNA expression in smallintestine, prostate, liver, and pancreas.

Measurement of SEQ ID NO:3; Clone ID 1341701; Gene ID 29634 (Cln106)

Absolute numbers are depicted in Table 2 as relative levels ofexpression of Cln106 (SEQ ID NO:3) in 12 normal different tissues. Allthe values are compared to normal testis (calibrator). These RNA samplesare commercially available pools, originated by pooling samples of aparticular tissue from different individuals.

TABLE 2 Relative levels of Cln106 Expression in Pooled Samples TissueNORMAL Colon-Ascending 110 Endometrium 0 Kidney 0 Liver 0 Ovary 0Pancreas 0 Prostate 16 Small Intestine 0 Spleen 0 Stomach 0 Testis 1Uterus 0

The relative levels of expression in Table 2 show for the CSG Cln106(SEQ ID NO:3), mRNA expression is more than 6 fold higher in the pool ofnormal ascending colon (110) compared with prostate (16). Testis, thecalibrator, with a relative expression level of 1, is the only othertissue expressing the mRNA for Cln106 (SEQ ID NO:3). These resultsdemonstrate that mRNA expression of this CSG is highly specific forcolon.

The absolute numbers in Table 2 were obtained analyzing pools of samplesof a particular tissue from different individuals. They can not becompared to the absolute numbers originated from RNA obtained fromtissue samples of a single individual in Table 3.

The absolute numbers in Table 3 are relative levels of expression ofCln106 (SEQ ID NO:3) in 57 pairs of matching samples. All the values arecompared to normal testis (calibrator). A matching pair is formed bymRNA from the cancer sample for a particular tissue and mRNA from thenormal adjacent sample for that same tissue from the same individual.

TABLE 3 Relative levels of Cln106 Expression in Individual SamplesMatching Normal Sample ID Tissue Cancer Adjacent Sto AC93 Stomach 1 4 96Sto AC99 Stomach 2 0.4 0.5 Sml 21XA Small Intestine 1 0 0 Sml H89 SmallIntestine 2 0.93 1.28 Cln B56 Colon-Cecum (A) 1 317 101 Cln AS45Colon-Ascending (A) 2 316.3 146.5 Cln CM67 Colon-Cecum (B) 3 481.0 217.5Cln AS67 Colon-Ascending (B) 4 858.1 220.6 Cln AS43 Colon-Ascending (C)5 1370 98 Cln AS46 Colon-Ascending (C) 6 3051 375 Cln AS98Colon-Ascending (C) 7 26 42 Cln AS89 Colon-Ascending (D) 8 524.6 11.0Cln TX01 Colon-Transverse (B) 9 2886.3 1992.0 Cln TX89 Colon-Transverse(B) 10 146.0 35.9 Cln TX67 Colon-Transverse (C) 11 2.9 421.7 Cln MT38Colon-Splenic 1681 187 Flexture (M) 12 Cln SG89 Colon-Sigmoid (B) 131063.8 31.1 Cln SG67 Colon-Sigmoid (C) 14 8.5 9.4 Cln SG33 Colon-Sigmoid(C) 15 264 549 Cln SG45 Colon-Sigmoid (D) 16 580.0 114.6 Cln B34Colon-Rectosigmoid (A) 17 97 244 Cln CXGA Colon-Rectum (A) 18 45.1 273.4Cln RC67 Colon-Rectum (B) 19 2.7 20.0 Cln C9XR Colon-Rectosigmoid (C) 20609 460 Cln RS45 Colon-Rectosigmoid (C) 21 472.8 144.0 Cln RC01Colon-Rectum (C) 22 568 129 Cln RC89 Colon-Rectum (D) 23 4.6 322.91 Bld46XK Bladder 1 0.2 0 Bld 66X Bladder 2 1 1 Bld 32XK Bladder 3 0.0 0.0Kid 126XD Kidney 1 0 0 Kid 12XD Kidney 2 0 0 Kid 5XD Kidney 3 0.0 1.0Kid 6XD Kidney 4 0.0 0.0 Kid 106XD Kidney 5 0.4 0.0 Liv 42X Liver 1 0.00.0 Liv 15XA Liver 2 0.0 0.0 Liv 94XA Liver 3 0.0 0.0 Lng AC69 Lung 1 20 Lng BR94 Lung 2 0 0 Lng 47XQ Lung 3 0 0 Mam 59X Mammary Gland 1 0 0Mam B011X Mammary Gland 2 0 0 Mam A06X Mammary Gland 3 0 0 Ovr 103XOvary 1 0.04 2.08 Ovr 130X Ovary 2 0.1 2.76 Pan 71XL Pancreas 1 4.08 0.1Pan 82XP Pancreas 2 0 0 Pro 12B Prostate 1 0.3 0 Pro 23B Prostate 2 3 4Pro 13XB Prostate 3 2 7 Pro 34B Prostate 4 0.54 4.01 Pro 20XB Prostate 54.8 4.3 Pro 65XB Prostate 6 0.7 1.3 Tst 39X Testis 1 2.78 0 End 8XAEndometrium 1 0 0.2 Utr 85XU Uterus 1 1.26 0 0 = Negative

When matching samples were analyzed, the higher levels of expressionwere in the colon, showing a high degree of tissue specificity for thistissue. These results confirm the tissue specificity results obtainedwith the panel of normal pooled samples (Table 2). Furthermore, thelevel of mRNA expression in cancer samples and the isogenic normaladjacent tissue from the same individual were compared. This comparisonprovides an indication of specificity for the cancer stage (e.g. higherlevels of mRNA expression in the cancer sample compared to the normaladjacent). Table 3 shows overexpression of Cln106 (SEQ ID NO:3) in 15colon cancer tissues compared with their respective normal adjacent(colon samples #1, 2, 3, 4, 5, 6, 8, 9, 10, 12, 13, 16, 20, 21, and 22).There is overexpression in the cancer tissue for 65% of the colonmatching samples tested (total of 23 colon matching samples). Thematching sample Pan 71XL is a secondary cancer in pancreas, the primarycancer in that individual was a duodenal cancer.

Altogether, the high level of tissue specificity, plus the mRNAoverexpression in 65% of the colon matching samples tested aredemonstrative of CSG Cln106 (SEQ ID NO:3) being a diagnostic marker forcolon cancer.

Measurement of SEQ ID NO:4; Clone ID 816257; Gene ID 406452 (Cln107)

Absolute numbers as depicted in Table 4 are relative levels ofexpression of CSG Cln107 (SEQ ID NO:4) in 12 normal different tissues.All the values are compared to normal small intestine (calibrator).These RNA samples are commercially available pools, originated bypooling samples of a particular tissue from different individuals.

TABLE 4 Relative levels of Cln107 Expression in Pooled Samples TissueNORMAL Colon-Ascending 3.2 Endometrium 0 Kidney 0.2 Liver 0 Ovary 0Pancreas 0 Prostate 0.1 Small Intestine 1 Spleen 0 Stomach 0.3 Testis 0Uterus 0

The relative levels of expression in Table 4 show that mRNA expressionof the CSG Cln107 (SEQ ID NO:4) is more than 10 fold higher in the poolof normal ascending colon (3.2), five fold higher in small intestine(1), and 1.5 fold higher in stomach (0.3), compared with the next higherexpresser (0.2 for kidney). Seven of the pooled tissues samples analyzedwere negative and prostate showed a relative expression of 0.1 forCln107 (SEQ ID NO:4). These results demonstrate that Cln107 mRNAexpression is highly specific for colon, small intestine, and in a lowerdegree for stomach.

The absolute numbers in Table 4 were obtained analyzing pools of samplesof a particular tissue from different individuals. They can not becompared to the absolute numbers originated from RNA obtained fromtissue samples of a single individual in Table 5.

The absolute numbers in Table 5 are relative levels of expression ofCln107 (SEQ ID NO:4) in 57 pairs of matching samples. All the values arecompared to normal small intestine (calibrator). A matching pair isformed by mRNA from the cancer sample for a particular tissue and mRNAfrom the normal adjacent sample for that same tissue from the sameindividual.

TABLE 5 Relative levels of Cln107 Expression in Individual SamplesMatching Normal Sample ID Tissue Cancer Adjacent Sto AC93 Stomach 1 8.913.4 Sto AC99 Stomach 2 6.0 0.9 Sml 21XA Small Intestine 1 1.07 1.42 SmlH89 Small Intestine 2 0.97 4.13 Cln B56 Colon-Cecum (A) 1 2 16 Cln AS45Colon-Ascending (A) 2 0.7 2.1 Cln CM67 Colon-Cecum (B) 3 1.6 2.1 ClnAS67 Colon-Ascending (B) 4 1.2 6.2 Cln AS43 Colon-Ascending (C) 5 13.50.5 Cln AS46 Colon-Ascending (C) 6 9.7 23.6 Cln AS98 Colon-Ascending (C)7 28.1 1.4 Cln AS89 Colon-Ascending (D) 8 0.9 3.1 Cln TX01Colon-Transverse (B) 9 3.0 10.6 Cln TX89 Colon-Transverse (B) 10 4.5 0.6Cln TX67 Colon-Transverse (C) 11 3.6 3.4 Cln MT38 Colon-Splenic 4.0 2.6Flexture (M) 12 Cln SG89 Colon-Sigmoid (B) 13 4.7 0.9 Cln SG67Colon-Sigmoid (C) 14 1.0 1.3 Cln SG33 Colon-Sigmoid (C) 15 14.2 7.6 ClnSG45 Colon-Sigmoid (D) 16 4.8 6.0 Cln B34 Colon-Rectosigmoid (A) 17 3 2Cln CXGA Colon-Rectum (A) 18 4.4 1.9 Cln RC67 Colon-Rectum (B) 19 0.10.4 Cln C9XR Colon-Rectosigmoid (C) 20 5 3 Cln RS45 Colon-Rectosigmoid(C) 21 11.4 4.6 Cln RC01 Colon-Rectum (C) 22 1.8 2.3 Cln RC89Colon-Rectum (D) 23 0.1 5.35 Bld 46XK Bladder 1 0.2 0 Bld 66X Bladder 21 1 Bld 32XK Bladder 3 0.1 0.1 Kid 126XD Kidney 1 0 0.02 Kid 12XD Kidney2 0.1 0.2 Kid 5XD Kidney 3 0.3 0.0 Kid 6XD Kidney 4 0.1 0.1 Kid 106XDKidney 5 0.0 0.1 Liv 42X Liver 1 7.9 0.002 Liv 15XA Liver 2 0.0 0.0 Liv94XA Liver 3 0.0 0.0 Lng AC69 Lung 1 1.6 0.2 Lng BR94 Lung 2 0.4 0 Lng47XQ Lung 3 0.78 0.2 Mam 59X Mammary Gland 1 0.05 0.3 Mam B011X MammaryGland 2 0.01 0.004 Mam A06X Mammary Gland 3 0.22 0 Ovr 103X Ovary 1 0.010.01 Ovr 130X Ovary 2 0.09 0.1 Pan 71XL Pancreas 1 2.51 2.81 Pan 82XPPancreas 2 0 0.62 Pro 12B Prostate 1 0.3 0.1 Pro 23B Prostate 2 0.3 0.2Pro 13XB Prostate 3 0 0 Pro 34B Prostate 4 0.04 0.22 Pro 20XB Prostate 50.4 0.1 Pro 65XB Prostate 6 0.0 0.1 Tst 39X Testis 1 0.02 0.01 End 8XAEndometrium 1 0.01 0.5 Utr 85XU Uterus 1 0.03 0 0 = Negative

When matching samples were analyzed, the higher levels of expressionwere in colon, stomach, and small intestine, showing a high degree oftissue specificity for colon tissues. These results confirm the tissuespecificity results obtained with normal pooled samples (Table 4).Furthermore, the level of mRNA expression in cancer samples and theisogenic normal adjacent tissue from the same individual were compared.This comparison provides an indication of specificity for the cancerstage (e.g. higher levels of mRNA expression in the cancer samplecompared to the normal adjacent). Table 5 shows overexpression of Cln107(SEQ ID NO:4) in 11 colon cancer tissues compared with their respectivenormal adjacent (colon samples #5, 7, 10, 11, 12, 13, 15, 17, 18, 20,and 21). There is overexpression in the cancer tissue for 48% of thecolon matching samples tested (total of 23 colon matching samples). Thematching sample Pan 71XL is a secondary cancer in pancreas, the primarycancer in that individual was a duodenal cancer.

Altogether, the high level of tissue specificity, plus the mRNAoverexpression in almost half of the colon, stomach, and small intestinematching samples tested are demonstrative of CSG Cln107 (SEQ ID NO:4)being a diagnostic marker for colon cancer.

Measurement of SEQ ID NO:5; Clone ID 775133; Gene ID 24508 (Cln108)

The absolute numbers shown in Table 6 are relative levels of expressionof CSG Cln108 (SEQ ID NO:5) in 12 normal different tissues. All thevalues are compared to normal small intestine (calibrator). These RNAsamples are commercially available pools, originated by pooling samplesof a particular tissue from different individuals.

TABLE 6 Relative levels of Cln108 Expression in Pooled Samples TissueNORMAL Colon-Ascending 2846.5 Endometrium 1 Kidney 5.5 Liver 18.7 Ovary3.4 Pancreas 198.1 Prostate 1024 Small Intestine 810.8 Spleen 32.2Stomach 9981.2 Testis 0 Uterus 294.1The relative levels of expression in Table 6 show that mRNA expressionof CSG Cln108 (SEQ ID NO:5) is more than 10 fold higher in the pool ofnormal ascending colon (2846.5) and almost ten fold higher in stomach(9981.2), compared to the expression level in any other tissue analyzed.These results demonstrate that mRNA expression of this CSG is alsohighly specific for colon and stomach.

The absolute numbers in Table 6 were obtained analyzing pools of samplesof a particular tissue from different individuals. They can not becompared to the absolute numbers originated from RNA obtained fromtissue samples of a single individual in Table 7.

The absolute numbers depicted in Table 7 are relative levels ofexpression of Cln108 (SEQ ID NO:5) in 57 pairs of matching samples. Allthe values are compared to normal small intestine (calibrator). Amatching pair is formed by mRNA from the cancer sample for a particulartissue and mRNA from the normal adjacent sample for that same tissuefrom the same individual.

TABLE 7 Relative levels of Cln108 Expression in Individual SamplesMatching Normal Sample ID Tissue Cancer Adjacent Sto AC93 Stomach 128696 34842 Sto AC99 Stomach 2 21523 30862 Sml 21XA Small Intestine 12944 964.4 Sml H89 Small Intestine 2 244.5 3513.2 Cln B56 Colon-Cecum(A) 1 27242 24637 Cln AS45 Colon-Ascending (A) 2 5827.0 8771.0 Cln CM67Colon-Cecum (B) 3 4251.0 4684.0 Cln AS67 Colon-Ascending (B) 4 564.01949.0 Cln AS43 Colon-Ascending (C) 5 50310 10949 Cln AS46Colon-Ascending (C) 6 246044 120073 Cln AS98 Colon-Ascending (C) 7 4044217482 Cln AS89 Colon-Ascending (D) 8 5730.0 1581.0 Cln TX01Colon-Transverse (B) 9 22281.0 114784.0 Cln TX89 Colon-Transverse (B) 1011026.0 1639.0 Cln TX67 Colon-Transverse (C) 11 17004.0 11654.0 Cln MT38Colon-Splenic 77589 31620 Flexture (M) 12 Cln SG89 Colon-Sigmoid (B) 13140339.0 49617.0 Cln SG67 Colon-Sigmoid (C) 14 4951.0 7905.0 Cln SG33Colon-Sigmoid (C) 15 60875 120490 Cln SG45 Colon-Sigmoid (D) 16 30437.047267.0 Cln B34 Colon-Rectosigmoid (A) 17 5848 5861 Cln CXGAColon-Rectum (A) 18 13877.0 9787.0 Cln RC67 Colon-Rectum (B) 19 1703.026589.0 Cln C9XR Colon-Rectosigmoid (C) 20 2458 19071 Cln RS45Colon-Rectosigmoid (C) 21 95523 61939 Cln RC01 Colon-Rectum (C) 2298891.0 80047.0 Cln RC89 Colon-Rectum (D) 23 17.0 1775 Bld 46XK Bladder1 0 8 Bld 66X Bladder 2 397 44 Bld 32XK Bladder 3 0.0 16.0 Kid 126XDKidney 1 32 22 Kid 12XD Kidney 2 6 0 Kid 106XD Kidney 3 4.0 33.0 Liv 42XLiver 1 4783 0 Liv 15XA Liver 2 4.0 10.0 Liv 94XA Liver 3 159.0 21.0 LngAC69 Lung 1 222 295 Lng BR94 Lung 2 112 0 Lng 47XQ Lung 3 30 69 Lng AC66Lung 4 29 137 Mam 59X Mammary Gland 1 56 0 Mam B011X Mammary Gland 2 5431 Mam A06X Mammary Gland 3 12 0 Ovr 103X Ovary 1 37 0 Pan 71XL Pancreas1 13203 4163 Pan 82XP Pancreas 2 39.1 0 Pro 12B Prostate 1 386 88 Pro23B Prostate 2 250 23 Pro 13XB Prostate 3 92 731 Pro 34B Prostate 4 33.3265.7 Pro 20XB Prostate 5 454.6 1908.9 Pro 65XB Prostate 6 733.5 922.0End 8XA Endometrium 1 5 92 Utr 85XU Uterus 1 98.9 21.8 Utr 23XU Uterus 235.3 0 Utr 135XO Uterus 3 39.2 43.8 Utr 141XO Uterus 4 212.1 55.9 0 =Negative

When matching samples were analyzed, the higher levels of expressionwere in colon and stomach, showing a high degree of tissue specificityfor these two tissues. These results confirm the tissue specificityresults obtained with normal pooled samples (Table 6). Furthermore, thelevel of mRNA expression in cancer samples and the isogenic normaladjacent tissue from the same individual were compared. This comparisonprovides an indication of specificity for the cancer stage (e.g. higherlevels of mRNA expression in the cancer sample compared to the normaladjacent). Table 7 shows overexpression of CSG Cln108 (SEQ ID NO:5) in13 colon cancer tissues compared with their respective normal adjacent(colon samples #1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 18, 21, and 22). Thereis overexpression in the cancer tissue for 56% of the colon matchingsamples tested (total of 23 colon matching samples). The matching samplePan 71XL is a secondary cancer in pancreas, the primary cancer in thatindividual was a duodenal cancer.

Altogether, the high level of tissue specificity, plus the mRNAoverexpression in more than half of the colon, stomach, and smallintestine matching samples tested are demonstrative of this CSG, Cln108(SEQ ID NO:5), also being a diagnostic marker for colon cancer.

Measurement of SEQ ID NO:7; Clone ID 2348122; Gene ID 23371 (Cln109)

The absolute numbers depicted in Table 8 are relative levels ofexpression of CSG Cln109 (SEQ ID NO:7) in 12 normal different tissues.All the values are compared to normal ovary (calibrator). These RNAsamples are commercially available pools, originated by pooling samplesof a particular tissue from different individuals.

TABLE 8 Relative levels of Cln109 Expression in Pooled Samples TissueNORMAL Colon-Ascending 28.8 Endometrium 0.45 Kidney 0.41 Liver 0.72Ovary 0.07 Pancreas 82.8 Prostate 124.3 Small Intestine 626.4 Spleen 1.2Stomach 12.05 Testis 1.51 Uterus 52.99The relative levels of expression in Table 8 show that mRNA expressionof CSG Cln109 (SEQ ID NO:7), is more than 5 fold higher in the pool ofnormal small intestine (626.4) compared to the expression level in anyother tissue analyzed. These results demonstrate that Cln109 (SEQ IDNO:7) mRNA expression is highly specific for small intestine.

The absolute numbers in Table 8 were obtained analyzing pools of samplesof a particular tissue from different individuals. They can not becompared to the absolute numbers originated from RNA obtained fromtissue samples of a single individual in Table 9.

The absolute numbers depicted in Table 9 are relative levels ofexpression of Cln109 (SEQ ID NO:7) in 53 pairs of matching samples. Allthe values are compared to normal ovary (calibrator). A matching pair isformed by mRNA from the cancer sample for a particular tissue and mRNAfrom the normal adjacent sample for that same tissue from the sameindividual.

TABLE 9 Relative levels of Cln109 Expression in Individual SamplesMatching Normal Sample ID Tissue Cancer Adjacent Sto AC93 Stomach 1 25741310 Sto AC99 Stomach 2 4153 5 Sml 21XA Small Intestine 1 2667 13663.8Sml H89 Small Intestine 2 57.8 904.29 Cln B56 Colon-Cecum (A) 1 6794 299Cln AS45 Colon-Ascending (A) 2 814.6 105.8 Cln CM67 Colon-Cecum (B) 3294.6 36.1 Cln AS67 Colon-Ascending (B) 4 2.2 26.3 Cln AS43Colon-Ascending (C) 5 111 377 Cln AS46 Colon-Ascending (C) 6 1180 352Cln AS98 Colon-Ascending (C) 7 1075 92 Cln AS89 Colon-Ascending (D) 814022.7 87.5 Cln TX01 Colon-Transverse (B) 9 1027.6 282.1 Cln TX89Colon-Transverse (B) 10 2.5 23.7 Cln TX67 Colon-Transverse (C) 11 0.172.3 Cln MT38 Colon-Splenic 372 88 Flexture (M) 12 Cln SG89Colon-Sigmoid (B) 13 179.2 33.4 Cln SG67 Colon-Sigmoid (C) 14 85.0 94.7Cln SG33 Colon-Sigmoid (C) 15 5461 377 Cln SG45 Colon-Sigmoid (D) 16762.7 15.9 Cln B34 Colon-Rectosigmoid (A) 17 460 1 Cln RC67 Colon-Rectum(B) 18 64.5 136.2 Cln C9XR Colon-Rectosigmoid (C) 19 441 34 Cln RS45Colon-Rectosigmoid (C) 20 1931 195 Cln RC01 Colon-Rectum (C) 21 72.819.1 Cln RC89 Colon-Rectum (D) 22 4.8 90.2 Bld 46XK Bladder 1 4 3 Bld66X Bladder 2 1 0 Bld 32XK Bladder 3 0.1 307.6 Kid 126XD Kidney 1 0 2Kid 12XD Kidney 2 3 16 Kid 5XD Kidney 3 0.0 0.3 Kid 6XD Kidney 4 18.51.2 Liv 42X Liver 1 21 0.03 Liv 15XA Liver 2 0.5 0.4 Liv 94XA Liver 30.4 0.0 Lng AC69 Lung 1 0.1 0 Lng BR94 Lung 2 3 0 Lng 60XL Lung 3 0.1 0Mam 59X Mammary Gland 1 0 4 Mam B011X Mammary Gland 2 8 13 Mam A06XMammary Gland 3 4.7 9.6 Pan 71XL Pancreas 1 8902.5 1428.2 Pan 82XPPancreas 2 0.2 9.3 Pro 12B Prostate 1 9 20 Pro 23B Prostate 2 191 88 Pro13XB Prostate 3 12 460 Pro 34B Prostate 4 3.2 80.4 Tst 39X Testis 1 29.90 End 8XA Endometrium 1 0.3 21 Utr 85XU Uterus 1 244.7 592.2 Ovr 63AOvary 1 11.4 0 Ovr A1C Ovary 2 68.4 0 0 = Negative

When matching samples were analyzed, the higher levels of expressionwere in small intestine, colon and stomach, showing a high degree oftissue specificity for these three colon tissues. These results confirmthe tissue specificity results obtained with normal pooled samples forsmall intestine (Table 8). Furthermore, the level of mRNA expression incancer samples and the isogenic normal adjacent tissue from the sameindividual were compared. This comparison provides an indication ofspecificity for the cancer stage (e.g. higher levels of mRNA expressionin the cancer sample compared to the normal adjacent). Table 9 showsoverexpression of CSG, Cln109 (SEQ ID NO:7) in 15 colon cancer tissuescompared with their respective normal adjacent (colon samples #1, 2, 3,6, 7, 8, 9, 12, 13, 15, 16, 17, 19, 20, and 21). There is overexpressionin the cancer tissue for 68% of the colon matching samples tested (totalof 22 colon matching samples). The matching sample Pan 71XL is asecondary cancer in pancreas, the primary cancer in that individual wasa duodenal cancer.

Altogether, the high level of tissue specificity, plus the mRNAoverexpression in more than half of the colon, stomach, and smallintestine matching samples tested are demonstrative of CSG Cln109 (SEQID NO:7) being a diagnostic marker for colon cancer. The amino acidsequence encoded by the open reading frame of Cln109 is depicted in SEQID NO:10.

1. A method for detecting colon cancer in a patient comprising: (a) measuring levels of a polynucleotide comprising SEQ ID NO:3 or native protein encoded by SEQ ID NO:3 in a sample of cells, tissue or bodily fluid obtained from the patient; and (b) comparing the measured levels of the polynucleotide comprising SEQ ID NO:3 or the native protein encoded by SEQ ID NO:3 with levels of the polynucleotide comprising SEQ ID NO:3 or the native protein encoded by SEQ ID NO:3 in a sample of cells, tissue or bodily fluid obtained from a control, wherein an increase in measured levels of the polynucleotide comprising SEQ ID NO:3 or the native protein encoded by SEQ ID NO:3 in the patient versus the levels of the polynucleotide comprising SEQ ID NO:3 or the native protein encoded by SEQ ID NO:3 in the control is associated with the presence of colon cancer.
 2. The method of claim 1 wherein the sample is cells.
 3. The method of claim 1 wherein the sample is tissues.
 4. The method of claim 1 wherein the sample is bodily fluids.
 5. The method of claim 1 wherein levels of the polynucleotide comprising SEQ ID NO:3 are measured.
 6. The method of claim 5 wherein the sample is cells.
 7. The method of claim 5 wherein the sample is tissues.
 8. The method of claim 5 wherein the sample is bodily fluids.
 9. The method of claim 1 wherein levels of the native protein encoded by SEQ ID NO:3 are measured.
 10. The method of claim 9 wherein the sample is cells.
 11. The method of claim 9 wherein the sample is tissues.
 12. The method of claim 9 wherein the sample is bodily fluids. 